ABSTRACT BSTRACT In an effort to reduce the use of animals for toxicity testing, companies have developed bioengineered in vitro skin models to replace the Draize skin irritation test. The development of tissue models has been further pushed by the European Union that prohibited the use of animals in 2009 for collecting toxicological data on cosmetic ingredients. Supply of bioengineered tissues is a significant issue that can be solved by development of effective cryopreservation methods. Presently, tissues or skin constructs are made-to-order and require a lead time of several weeks before they can be used. A cryopreserved product would be available upon demand eliminating the order lead time required to manufacture the product. Additionally, quality control checks can be performed pre-shipment, not post-shipment as is the current practice due to the short product shelf life. Cryopreservation will permit economies of manufacturing scale, resulting in reduced bioengineered construct costs to both the manufacturers and the end users. The availability of cryopreserved human bioengineered epithelial constructs would also provide the end user greater flexibility for scheduling of experiments without concern regarding availability. Further, it would be possible to order from the same batch of product in order to have better control of uniformity within research studies. We have developed an ice-free vitrification method for cryopreservation of bioengineered skin constructs used in toxicity testing. In preliminary studies using ice-free vitrification, we have consistently demonstrated >80% viability of several types of human bioengineered epithelial constructs using ice-free vitrification that persists for several days post-rewarming. However, a limiting factor for preservation at larger scale in multiwell plates using current manual preservation methods is the inability to add and remove cryoprotectants fast enough to avoid cytotoxicity across an entire 24 well plate. In this Phase I proposal we plan to customize and integrate an automated liquid handling system to manage our current successful vitrification methods in one Specific Aim. As a result, we will demonstrate the ability to process four 24-well plates simultaneously in the same time frame as we presently process 4-6 constructs manually. The outcome will be measured by assessment of tissue viability and functional assays required by tissue suppliers for quality control. We will also track accuracy of the automated system by measuring volumes of solutions, plate and solution temperature, and plate processing time. The outcome of this proposal will be inline with our Company goal of commercializing our vitrification process by storing on- demand human bioengineered tissues for distribution to customers for drug or chemical screening and research applications.